Multifaceted regulation of siderophore synthesis by multiple regulatory systems in Shewanella oneidensis

Siderophore-dependent iron uptake is a mechanism by which microorganisms scavenge and utilize iron for their survival, growth, and many specialized activities, such as pathogenicity. The siderophore biosynthetic system PubABC in Shewanella can synthesize a series of distinct siderophores, yet how it is regulated in response to iron availability remains largely unexplored. Here, by whole genome screening we identify TCS components histidine kinase (HK) BarA and response regulator (RR) SsoR as positive regulators of siderophore biosynthesis. While BarA partners with UvrY to mediate expression of pubABC post-transcriptionally via the Csr regulatory cascade, SsoR is an atypical orphan RR of the OmpR/PhoB subfamily that activates transcription in a phosphorylation-independent manner. By combining structural analysis and molecular dynamics simulations, we observe conformational changes in OmpR/PhoB-like RRs that illustrate the impact of phosphorylation on dynamic properties, and that SsoR is locked in the ‘phosphorylated’ state found in phosphorylation-dependent counterparts of the same subfamily. Furthermore, we show that iron homeostasis global regulator Fur, in addition to mediating transcription of its own regulon, acts as the sensor of iron starvation to increase SsoR production when needed. Overall, this study delineates an intricate, multi-tiered transcriptional and post-transcriptional regulatory network that governs siderophore biosynthesis.


Description of Supplementary Movie 1
The video depicts the 180-280 ns trajectory of VbrR D-RD-D51N , showing the switch residue as it transitions through four states.The REC domain was represented in green cartoon form, while the switch residue was illustrated as cyan sticks, and T99 was shown in cyan lines, with the α4-helix concealed.Furthermore, the distance between the CZ atom of the switch residue and the N atom in T99's backbone, as well as the diameter of the benzene ring, were measured.

Supplementary Figure 2 .
The siderophore production of mutants and their complementary strains used in the study.Expression of genes was driven by IPTG-inducible promoter Ptac with IPTG at varying concentrations.

Supplementary Figure 3 .Supplementary Figure 4 .
BarA-UvrY TCS and SsoR.BarA is known to phosphorylate UvrY via phospho-relay shown by arrowed lines.BarA may also possibly phosphorylate SsoR by arrowed dash line.HAMP, HAMP signal transduction domain; HisKa, histidine kinase dimerization domain; HATPase, histidine kinase ATPase domain; Rec, receiver domain; Hpt, histidine-containing phosphotransfer domain; wHTH, winged helix-turn-helix DNA-binding motif; HTH, helix-turn-helix DNA-binding motif.D52/54: 4-aspartylphosphate.The regulation of BarA/UvrY/Csr system on siderophore synthesis.a The growth of csrA strains in LB liquid and agar plates.Expression of csrA was driven by IPTG-inducible promoter Ptac with 0.1 mM IPTG.b The mRNA level of pubA and protein level in indicated strains carrying csr genes (under the control of tac promoters) were respectively measured.c The activity of arcA promoters are constitutive in the indicated strains.

Supplementary Figure 5 .Supplementary Figure 6 .Supplementary Figure 7 .Supplementary Figure 8 .
Phylogenetic tree and genomic backgrounds of RRs.On left, the phylogenetic tree shown in Figure5Ais given with the full name of the bacterial species.On right, the genomic backgrounds of the genes of interest, with RR genes in red boxes and kinase genes in black boxes.Q87HP4 VbrRVibrio parahaemolyticus 407 Structural similarity dendrogram and alignment results for the AlphaFoldpredicted and Crystal structures of E. coli PhoBs.a The dendrogram is calculated using DALI and derived by average linkage clustering of the structural similarity matrix (Dali Z-scores).The REC domains on the dendrogram include 12 monomers from five crystal structures of E. coli PhoB (highlighted in blue), as well as 52 monomers (with protein sequences identical to E. coli PhoB) from the AlphaFold Protein Structure Database.The proteins from the AlphaFold Protein Structure Database are distinguished by the states using red, purple, and green background.b and c show alignment results for the AlphaFold-predicted structure and Crystal structures, respectively.Expression and purification of proteins.His 6 -tagged recombinant proteins were expressed and purified from E. coli BL21(DE3).The purification conditions for Fur protein are natural, whereas the purification buffers for SsoR proteins are all denaturantadded.After induction with 0.2 mM IPTG overnight, cells were collected, disrupted by a French pressure cell disrupter, and debris was removed by centrifugation.The supernatant solution was loaded on a 5-ml HisTrap HP, and fractions were examined by SDS-PAGE and Coomassie brilliant blue staining.To renature the SsoR proteins, the eluted fractions containing SsoR proteins were diluted into 2 M urea, 20 mM Tris/HCl (pH 7.0), 1 mM EDTA by sequential dilutions and then dialyzed against 20 mM Tris/HCl (pH 7.0) overnight.All uncropped blots

Supplementary Table 1: TCSs in S. oneidensis
The transition between intermediate state A and intermediate state B, as well as from intermediate state B to the outer state, is accompanied by the compression of the benzene ring of the switching molecule.Moreover, the benzene ring is compressed and flipped when in the outer state, suggesting that this state becomes unstable after dimerization.